The long-term goal of this proposal it to enhance our understanding of acne, in particular the bacteriophages that infect and kill Propionibacterium acnes - a bacterium that contributes to the pathogenesis of the skin disease acne towards the development an effective phage-based antimicrobial therapy for the treatment of acne. Only a small number of P. acnes phages have been studied in any detail, and they display a number of interesting features. In particular, these phages are able to enter into a stable relationship with the host bacterium, known as a lysogen; however, their genomes lack all proteins known to be involved in the process of forming and maintaining a lysogenic state. It is proposed, therefore, that by sequencing and characterizing additional P. acnes phages, we will be able to learn more about the potentially novel way in which they lysogenize their host bacteria. A thorough understanding of this process will be critically important in the eventual choice of phages to be employed for therapeutic purposes, as functions that promote lysogeny must be avoided in any phage-based treatments. The first aim of this project will be to isolate P. acnes bacteriophages from the skin of acne patients and normal donors and to characterize their diversity in terms of propensity for lytic vs. lysogenic growth, as well as to determine the patterns of immunity conferred by the lysogenic phages. The last part of this aim will involve the development of a system for the genetic engineering P. acnes phages. This system will be utilized to perform the functional genomic studies outlined in the second part of this proposal and will ultimately be useful for the engineering of optimally virulent therapeutic phages. The second set of experiments will focus on elucidating the potentially novel mechanism(s) by which P. acnes bacteriophages are able to lysogenize their hosts. This will first involve performing a careful comparative genomic analysis of the phages sequenced in Aim 1, with a focus on genes that show variability in lytic and in lysogenic phages displaying differing immunity profiles. Once candidate genes are identified, these will be targeted for mutagenesis using the genetic tools developed in Aim 1, and the phenotypes of the mutant phages will be determined with regards to lysogeny and immunity. Genes that appear to be involved in these processes will be cloned for expression in P. acnes, to assay their function in vivo, and in Escherichia coli, for purification and use in biochemical assays, such as those to determine DNA binding, a feature of many proteins involved in the lysogenic pathway. Ultimately, these studies will provide new insight into the mechanisms by which bacteriophages derived from skin interact with their host bacteria, P. acnes, relevant to our understanding of, and potential therapy for acne.